Biochemistry 1989, 28, 6 157-6 160
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Effect of Antineoplastic Agents on the DNA Cleavage/Religation Reaction of Eukaryotic Topoisomerase 11: Inhibition of DNA Religation by Etoposidet Neil Osheroff Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-01 46 Received April 28, 1989; Revised Manuscript Received May 24, 1989
ABSTRACT: Beyond its essential physiological functions, topoisomerase I1 is the primary cellular target for
a number of clinically relevant antineoplastic drugs. Although the chemotherapeutic efficacies of these drugs correlate with their abilities to stabilize the covalent topoisomerase 11-DNA cleavage complex, their molecular mechanism of action has yet to be described. In order to characterize the drug-induced stabilization of this e n z y m e D N A complex, the effect of etoposide on the D N A cleavage/religation reaction of Drosophila melanogaster topoisomerase I1 was studied. Under the conditions employed, etoposide increased levels of enzyme-mediated double-stranded D N A cleavage 5-6-fold and single-stranded cleavage -4-fold. Maximal stimulation was observed a t 80-100 pM etoposide with 50% of the maximal effect a t 15 pM drug. By employing a topoisomerase I1 mediated D N A religation assay [Osheroff, N., & Zechiedrich, E. L. (1987) Biochemistry 26,4303-43091, etoposide was found to stabilize the enzyme-DNA cleavage complex (at least in part) by inhibiting the enzyme’s ability to religate cleaved D N A . Moreover, in order for the drug to affect religation, it had to be present a t the time of D N A cleavage.
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E u k a r y o t i c topoisomerase II is an essential enzyme which is involved in a number of fundamental cellular processes (Vosberg, 1985; Wang, 1985; Osheroff, 1989). Central to the physiological function of the enzyme is its ability to create and religate double-stranded breaks in the backbone of DNA (Vosberg, 1985; Wang, 1985; Osheroff, 1989). Beyond its normal cellular activities, topoisomerase I1 is the primary cellular target for a number of clinically relevant antineoplastic drugs (Zwelling, 1985; Glisson & Ross, 1987; Bodley & Liu, 1988). The chemotherapeutic efficacies of these agents correlate with their abilities to stabilize the covalent topoisomerase 11-DNA cleavage complex (Zwelling, 1985; Glisson & Ross, 1987; Bodley & Liu, 1988), which is an intermediate in the enzyme’s DNA strand passage reaction (Osheroff, 1989; Gale & Osheroff, 1989). Thus, as monitored in vitro, these drugs increase levels of enzyme-mediated DNA cleavage (Zwelling, 1985; Glisson & Ross, 1987; Bodley & Liu, 1988). Unfortunately, despite the important role of topoisomerase I1 in the treatment of human cancers, virtually nothing is known about the molecular mechanism by which antineoplastic drugs alter enzyme function. One of the major stumbling blocks that has prevented the detailed mechanism of drug action on topoisomerase I1 from being delineated has been the inability to uncouple the enzyme’s forward DNA cleavage reaction from the reverse religation event. Therefore, while it is possible to demonstrate that antineoplastic agents stimulate enzyme-mediated DNA breakage by shifting the cleavage/religation equilibrium toward cleavage, it is impossible to state with certainty whether these drugs act by enhancing the forward cleavage reaction or by inhibiting DNA religation. Recently, an assay was developed which uncouples topoisomerase I1 mediated religation from DNA cleavage (Osheroff & Zechiedrich, 1987). The assay takes advantage of the finding that DNA cleavage intermediates generated by topoisomerase I1 in the presence of calcium can be trapped in This work was supported by Grant GM-33944 from the National Institutes of Health.
0006-2960/89/0428-6 157$01.50/0
the absence of a protein denaturant (Osheroff & Zechiedrich, 1987). The resulting covalent enzymeDNA cleavage complex is kinetically competent and provides a substrate for which religation can be examined in the absence of the forward cleavage reaction. The above assay system has been employed to examine the mechanism by which etoposide, a potent antineoplastic drug (Issell, 1982; Issell et al., 1984; Muggia & McVie, 1987), stabilizes the cleavage complex between DNA and Drosophila melanogaster topoisomerase 11. Results indicate that the drug acts (at least in part) by inhibiting the enzyme-mediated religation of cleaved DNA. In addition, etoposide has to be present at the time of DNA cleavage in order to affect religation. A preliminary report of this work has appeared (Osheroff & Gale, 1988). EXPERIMENTAL PROCEDURES DNA topoisomerase I1 was purified from the nuclei of D . melanogaster Kc tissue culture cells as described by Shelton et al. (1983). Negatively supercoiled bacterial plasmid pBR322 (Bolivar et al., 1977) DNA was isolated from Escherichia coli DHl by a Triton X-100 lysis procedure followed by a double banding in cesium chloride-ethidium bromide gradients (Maniatis et al., 1982). Etoposide (VePesid, VP16-23) was obtained from Bristol Laboratories as a sterile 20 mg/mL solution in etoposide diluent [2 mg/mL citric acid, 30 mg/mL benzyl alcohol, 80 mg/mL polysorbate 80/Tween 80, 650 mg/mL poly(ethy1ene glycol) 300, 30.5% (v/v) ethanol]. The drug was stored at room temperature as per the manufacturer’s instructions. Tris and ethidium bromide were obtained from Sigma; analytical reagent grade CaC12.H20and MgCl2.6H20 were from Fisher; and sodium dodecyl sulfate (SDS)’ and proteinase K were from E. Merck Biochemicals. All other chemicals were of analytical reagent grade. I Abbreviations: SDS, sodium dodecyl sulfate; Tris-HC1, tris(hydroxymethy1)aminomethane hydrochloride; EDTA, ethylenediaminetetraacetic acid.
0 1989 American Chemical Society
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6158 Biochemistry, Vol. 28, No. 15, 1989 Cleavage of DNA by Topoisomerase I I . DNA cleavage reactions employed 75 nM topoisomerase I1 and 5 nM (0.3 pg) negatively supercoiled pBR322 DNA in a total of 20 pL of cleavage buffer (10 mM Tris, pH 7.9, 50 mM NaC1, 50 mM KCI, 0.1 mM EDTA, and 15 pg/mL bovine serum albumin) which contained 5 mM MgC1,. Samples were incubated at 30 OC for 6 min. DNA cleavage products were trapped by the addition of 2 pL of 10% SDS. One microliter of 250 mM EDTA and 2 pL of 0.8 mg/mL proteinase K were added and mixtures were incubated at 45 "C for 30 min to digest the topoisomerase 11. Products were mixed with 3 p L of loading buffer (60% sucrose, 0.05% bromophenol blue, 0.05% xylene cyanole FF, and 10 mM Tris-HCI, pH 7.9), heated at 75 OC for 2 min, and subjected to electrophoresis in 1 .O% agarose (MCB) gels in 40 mM Tris-acetate, pH 8.3, and 2 mM EDTA at 5 V/cm. Following electrophoresis, gels were stained in an aqueous solution of ethidium bromide (1 pg/mL). DNA bands were visualized by transillumination with ultraviolet light (300 nm) and photographed through Kodak Nos. 24A and 12 filters with Polaroid type 665 positive/negative film. The amount of DNA was quantitated by scanning negatives with a Biomed Instruments Model SL504-XL scanning densitometer. Under the conditions employed, the intensity of the negative was directly proportional to the amount of DNA present. The effect of etoposide on topoisomerase I1 mediated DNA cleavage was examined over a range of 0-100 pM drug. Control samples always contained an amount of etoposide diluent equivalent to that in the drug sample. Over the drug concentration range employed, topoisomerase I1 activity was not affected by the diluent. Religation of Cleaved DNA by Topoisomerase I I . The religation assay of Osheroff and Zechiedrich (1987) was employed. Topoisomerase I1 (75 nM) and 5 nM negatively supercoiled pBR322 DNA were incubated for 6 min at 30 OC in 20 pL of cleavage buffer which contained 5 mM CaCl,. Kinetically competent covalent topoisomerase II-DNA cleavage complexes were trapped by the addition of 2 pL of 100 mM EDTA. NaCl (0.8 p L of a 5 M solution) was added to prevent recleavage of DNA during the religation reaction (Liu et al., 1983; Osheroff & Zechiedrich, 1987). Samples were placed on ice to slow reaction rates, and religation was initiated by the addition of cold MgC12 (7.5 mM final concentration). SDS (2 pL of a 10% solution) was added to terminate religation at various time points up to 30 s. Samples were digested with proteinase K and were analyzed by electrophoresis as described above. The effect of antineoplastic agents on the topoisomerase I1 mediated DNA religation reaction was determined by including 100 pM etoposide in assays prior to the trapping of enzyme-DNA cleavage complexes. As above, control samples contained an equivalent amount of etoposide diluent equivalent to that in the drug sample. Rates of enzyme-mediated DNA religation were not affected by the diluent.
RESULTS Effect of Etoposide on the DNA Cleavage/Religation Equilibrium of Drosophila Topoisomerase II. In order to simplify the analysis of etoposide's action on topoisomerase I1 mediated DNA cleavage/religation, assays were carried out in the absence of ATP. Thus, reactions presented below represent the cleavage and religation events which take place prior to the enzyme's catalysis of DNA strand passage (Hsieh & Brutlag, 1980; Osheroff et al., 1983; Osheroff, 1989). As previously demonstrated for mammalian topoisomerase I1 (Chen et al., 1984; Ross et al., 1984; Yang et al., 1985),
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1: Effect of etoposide on the DNA cleavage/religation equilibrium of D. melanogaster topoisomerase 11. Assays were carried out as described under Experimental Procedures. The top panel shows reaction products that were subjected to electrophoresis on a 1% agarose gel. DNA standards (Std) are shown in the first two lanes. The concentration of etoposide included in assay mixtures is indicated at the top. The positions of fully supercoiled DNA (form I, FI), nicked circular plasmid molecules (form 11, FII), and linear molecules (form 111, FIII) are indicated. Results are depicted graphically in the bottom panel. The effects of etoposide on the production of double-stranded (DS) and single-stranded ( S S ) DNA breaks by topoisomerase I1 are shown. FIGURE
etoposide had a profound influence on the DNA cleavage/ religation equilibrium of the type I1 enzyme from D. melanogaster. Under the conditions employed, the drug shifted the equilibrium markedly toward cleavage, enhancing the formation of enzyme-mediated double-stranded breaks by 5-6-fold and single-stranded breaks by approximately 4-fold (Figure 1). DNA breakage (both double and single stranded) was enhanced 50% at approximately 15 pM etoposide and maximal enhancement was observed at 80-100 p M drug. These values approximate the physiological drug concentrations found in the plasma of human patients immediately following acute chemotherapeutic treatment (Creaven, 1984). All of the effects of etoposide on DNA were mediated through topoisomerase 11. To this point, linear (i.e., doubly cut) and nicked (i.e., singly cut) DNAs generated in the presence of the drug were covalently attached to topoisomerase I1 (not shown). Cleaved nucleic acids were released from the enzyme only after digestion of the cleavage complex with proteinase K. The covalent linkage of the enzyme to its DNA products is a hallmark of topoisomerase-mediated cleavage (Sander & Hsieh, 1983; Liu et al., 1983; Osheroff & Zechiedrich, 1987). Additionally, in the presence of 100 pM etoposide, no linear or nicked DNA was produced in the absence of topoisomerase I1 (not shown). EDTA Reversibility of Topoisomerase 11 Generated DNA Breaks Formed in the Presence of Etoposide. The topoisomerase TI mediated DNA cleavage reaction (carried out in the presence of magnesium) is readily reversed by the addition of salt or EDTA (Sander & Hsieh, 1983; Liu et al., 1983; Osheroff & Zechiedrich, 1987). This is due primarily to the fact that topoisomerase I1 never releases its cleaved DNA intermediate (Vosberg, 1985; Wang, 1985; Osheroff, 1989). Enzyme-generated DNA breaks formed in the presence
Biochemistry, Vol. 28, No. 15, 1989
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1 2 3 4 EDTA reversibility of DNA breaks mediated by topoisomerase I I in the presence of 100 pM etoposide. Assays were carried out as described under Experimental Procedures. Lanes 1 and 2, DNA standards; lane 3, SDS was added prior to EDTA; lane 4,EDTA was added prior to SDS. FIGURE 2:
Znd STRAND
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Time
I
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Effect of etoposide on the DNA religation reaction of Drosophila topoisomerase 11. A semilogrithmic plot of percent DNA versus time is shown. The religation of the first DNA strand (Le., the conversion of linear DNA to nicked circular molecules) is displayed in the top panel. Results are plotted as the loss of linear DNA. The religation of the second DNA strand (i.e., the conversion of nicked circular DNA to negatively supercoiled molecules) is displayed in the bottom panel. Results are plotted as the difference between the percent negatively supercoiled DNA at the completion of the reaction (at t,, FI DNA = 100%)and the percent negatively supercoiled DNA at any reaction time ( t ) . The open circles depict reactions that contained no drug; the closed circles depict reactions in which 100 pM etoposide was present at the time of enzyme-mediated DNA cleavage and religation; the closed squares depict reactions in which no drug was present at the time the enzymeDNA cleavage complex was trapped, but 100 p M etoposide was added prior to the initiation of religation (Le., immediately after the EDTA). FIGURE 4:
1 2 3 4 5 6 DNA religation reaction of Drosophila topoisomerase 11. Assays were carried out as described under Experimental Procedures. Lanes 1 and 2, DNA standards; lane 3, religation of DNA at zero time; lane 4,religation of DNA at 1 min following the addition of magnesium; lanes 5 and 6, assays (in the absence or presence of 100 p M etoposide, respectively) in which topoisomerase I1 was left out of reaction mixtures through step 3 of the assay (see text) and was added immediately prior to the addition of magnesium. FIGURE 3:
of etoposide and mammalian topoisomerase I1 have also been shown to be reversed (i.e., religated) following the addition of 0.5 M sodium chloride to reaction mixtures (Chen et al., 1984). In order to determine whether the etoposide-enhanced DNA breakage observed with Drosophila topoisomerase I1 was also reversible, the experiment shown in Figure 2 was carried out. I n this experiment, the Drosophila enzyme was allowed to establish a DNA cleavage/religation equilibrium in the presence of 100 p M etoposide and 5 mM magnesium, but the divalent cation was chelated with 10 mM (final concentration) EDTA prior to the addition of SDS. As seen in lane 4, drug-enhanced double- and single-stranded DNA breaks were reversed by this procedure (compare with lane 3, in which SDS was added prior to EDTA). Taken together with the previous study on salt reversibility (Chen et al., 1984), this result demonstrates that etoposide acts by stabilizing the active topoisomerase I1 cleaved DNA intermediate rather than by trapping it in a “dead-end” complex. This leads to the simple conclusion that etoposide must enhance the level of the cleavage intermediate either by stimulating the forward rate of enzyme-mediated DNA cleavage or by inhibiting the rate of enzyme-mediated religation (or both). Inhibition of Topoisomerase II Mediated DNA Religation by Etoposide. As a direct test of the above conclusion, the effect of etoposide on the rate of topoisomerase I1 mediated religation of cleaved DNA was determined. For this purpose, the DNA religation assay described by Osheroff and Zechiedrich ( 1 987) was employed. This assay, which is shown in Figure 3, takes advantage of the fact that (unlike magnesium) calcium can be used to trap a kinetically competent covalent topoisomerase 11-DNA cleavage intermediate. It proceeds as follows. (1) In order to establish a DNA cleavage/religation equilibrium, topoisomerase I1 was incubated with negatively supercoiled pBR322 DNA in the presence of
5 mM calcium and 100 mM salt at 30 “ C . (2) EDTA (10 mM final concentration) was added to chelate the calcium and trap the active enzyme-DNA cleavage complex (Osheroff & Zechiedrich, 1987) (lane 3). (3) The salt concentration was increased from 100 to 250 mM to prevent recleavage of the DNA during the course of religation (Liu et al., 1983; Osheroff & Zechiedrich, 1987). (4) Religation was initiated on ice (to slow reaction rates) by the addition of excess magnesium ions. A 1-min time point is shown in lane 4. A previous kinetic analysis of this assay (Zechiedrich et al., 1989) revealed that topoisomerase I1 religates cleaved DNA via a sequential two-step mechanism. Linear (i.e., doubly cut) DNA is first converted to nicked (i.e., singly cut) DNA which in turn is converted to the original negatively supercoiled substrate. The first-order rate constant for the first religation step is approximately 6-fold faster than that of the second. In order to demonstrate that religated DNA was not being recleaved during the course of the assay, topoisomerase I1 was left out of reaction mixtures through step 3 of the assay. The enzyme was added immediately before the addition of magnesium to see if DNA cleavage was possible under the final reaction conditions. As seen in Figure 3, no DNA cleavage was observed under the final conditions for religation, in either the absence (lane 5) or presence (lane 6) of 100 pM etoposide. With the assay described above, 100 pM etoposide was found to inhibit the topoisomerase I1 mediated religation of cleaved DNA. Results are presented as a semilogarithmic plot of percent DNA versus time (Figure 4). This type of plot was employed because it allows the direct quantitation of apparent rate constants for a first-order process (Segel, 1975; Fersht, 1985). Religation of the first strand of DNA (Le., FIII FII) is shown in the top panel, while religation of the second
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strand (Le., FII FI) is shown in the bottom panel. When drug was present at the time of DNA cleavage, rates of religation for both DNA strands were inhibited approximately 3-fold (closed circles). Etoposide lowered the apparent first-order rate constant for the religation of the first strand from 0.20 to 0.07 s-l and that for the religation of the second strand from 0.03 to 0.01 s-’. Finally, in order for etoposide to inhibit enzyme-mediated religation, it had to be present at the time of DNA cleavage. This conclusion is based on the following experiment. Rather than adding the drug to reaction mixtures prior to trapping the topoisomerase 11-DNA cleavage complex with EDTA, 100 FM etoposide was added immediately after the chelator. Subsequent to the addition of salt and magnesium, the rate of DNA religation (Figure 4, closed squares) was indistinguishable from that observed in the absence of drug (open circles). DISCUSSION Understanding the molecular mechanism by which a drug mediates its action is critical to rational drug design. Although the mechanism(s) of topoisomerase I1 targeted drugs has yet to be determined, it has long been assumed that these antineoplastic agents stabilize enzyme-DNA cleavage complexes by interfering with the ability of topoisomerase I1 to religate its bound DNA intermediates. The data presented above validate this assumption. Indeed, etoposide does inhibit the DNA religation reaction of Drosophila topoisomerase 11. It should be noted, however, that the drug-induced 3-fold inhibition of DNA religation is somewhat less than its 5-6-fold enhancement of double-stranded DNA breakage. Since the experimental conditions (i.e., temperature, ionic strength, and divalent cation concentration) employed for the DNA religation assay differed from those utilized in cleavage reactions, it is not necessarily possible to directly compare results generated by the two systems. Thus, while it is clear that etoposide inhibits topoisomerase 11 mediated religation, it is not yet definitive that this represents its sole mode of action. It is quite possible that etoposide increases levels of enzyme-DNA cleavage intermediates by stimulating the forward rate of DNA cleavage in addition to its inhibitory effects on religation. Recent studies indicate that etoposide may be able to bind to both DNA (Chow et al., 1988) and topoisomerase I1 (Osheroff & Gale, 1988). However, the site of drug action has yet to be determined. Since etoposide had to be present at the time of DNA cleavage to inhibit religation, it is tempting to speculate that the drug acts within the enzyme-DNA ternary complex. Hopefully, future studies will be able to address this important mechanistic point. Finally, in a study similar to that presented above, the topoisomerase I1 targeted chemotherapeutic drug m-AMSA (Nelson et al., 1984; Tewey et al., 1984; Pommier et al., 1984, 1985) was also found to inhibit the DNA religation reaction of the Drosophila enzyme (M. J. Robinson & N . Osheroff, unpublished results). This finding suggests that even structurally disparate classes of antineoplastic agents may alter topoisomerase I1 function through a similar mechanism of action. ACKNOWLEDGMENTS I thank E. L. Zechiedrich for her critical reading of the manuscript, L. Smith for her assistance in preparing the topoisomerase I1 employed for this study, and S. Heaver for her conscientious preparation of the manuscript. Registry No. 80449-01-0.
Etoposide, 33419-42-0; DNA topoisomerase,
Accelerated Publications REFERENCES Bodley, A. L., & Liu, L. F. (1988) Biotechnology 6 , 1315-1319. Bolivar, F., Rodriquez, R., Greene, P. J., Betlach, M., Heyneker, H. L., Boyer, H. W., Crosa, J., & Falkow, S. (1977) Gene 2, 95-1 13. Chen, G. L., Yang, L., Rowe, T. C., Halligan, B. D., Tewey, K. M., & Liu, L. F. (1984) J . Biol. Chem. 259, 13560-13566. Chow, K.-C., Macdonald, T. L., & Ross, W. E. (1988) Mol. Pharmacol. 34, 467-473. Creaven, P. J. (1984) in Etoposide (VP-16): Current Status and New Development (Issell, B. F., Muggia, F. M., & Carter, S. K., Eds.) pp 103-115, Academic Press, New York. Fersht, A. (1 985) Enzyme Structure and Mechanism, 2nd ed., W. H. Freeman, New York. Gale, K. C., & Osheroff, N. (1989) J. Cell. Biochem., Suppf. 130, 87, Abstract L124. Glisson, B. S., & Ross, W. E. (1987) Pharmacol. Ther. 32, 89-106. Hsieh, T.-S., & Brutlag, D. (1980) Cell 21, 115-125. Issell, B. F. (1982) Cancer Chemother. Pharmacol. 7,73-80. Issell, B. F., Rudolph, A. R., & Louie, A. C. (1984) in Etoposide (VP-16): Current Status and New Development (Issell, B. F., Muggia, F. M., & Carter, S. K., Eds.) pp 1-11, Academic Press, New York. Liu, L. F., Rowe, T. C., Yang, L., Tewey, K. M., & Chen, G. L. (1983) J . Biol. Chem. 258, 15365-15370. Maniatis, T., Fritsch, E. F., & Sambrook, J. (1982) Molecular Cloning-A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. Muggia, F. M., & McVie, G. (1987) Natl. Cancer Inst. Monogr. 4, 129-133. Nelson, E. M., Tewey, K. M., & Liu, L. F. (1984) Proc. Natf. Acad. Sci. U.S.A.81, 1361-1365. Osheroff, N. (1989) Pharmacol. Ther. 41, 223-241. Osheroff, N., & Zechiedrich, E. L. (1 987) Biochemistry 26, 4303-4309. Osheroff, N., & Gale, K. C. (1988) FASEB J. 2, A1761, Abstract 8508. Osheroff, N., Shelton, E. R., & Brutlag, D. L. (1983) J . Biol. Chem. 258, 9536-9543. Pommier, Y., Schwartz, R. E., Kohn, K. W., & Zwelling, L. A. (1984) Biochemistry 23, 3194-3201. Pommier, Y., Schwartz, R. E., Zwelling, L. A., & Kohn, K. W. (1985) Biochemistry 24, 6406-6410. Ross, W., Rowe, T., Glisson, B., Yalowich, J., & Liu, L. F. (1984) Cancer Res. 44, 5857-5860. Sander, M., & Hsieh, T . S . (1983) J . Biol. Chem. 258, 842 1-8428. Segel, I. H. (1 975) Enzyme Kinetics, Wiley, New York. Shelton, E. R., Osheroff, N., & Brutlag, D. L. (1983) J . Biol. Chem. 258, 9530-9535. Tewey, K. M., Chen, G. L., Nelson, E. M., & Liu, L. F. (1984) J . Biof. Chem. 259, 9182-9187. Vosberg, H.-P. (1985) Curr. Top. Microbiol. Immunol. 114, 19-102. Wang, J . C. (1985) Annu. Rev. Biochem. 54, 665-697. Yang, L., Rowe, T. C., & Liu, L. F. (1985) Cancer Res. 45, 5872-5876. Zechiedrich, E. L., Christiansen, K., Andersen, A. H., Westergaard, O., & Osheroff, N. (1989) Biochemistry (in press). Zelling, L. A. (1985) Cancer Metastasis Rev. 4 , 263-276.
